Akt/PKB signaling plays a central role in many biological functions, such as cell proliferation, apoptosis, cell metabolism, and protein translation. Although it is well known that Akt activation is regulated by phosphorylation, it is unclear whether other types of the posttranslational modifications, such as ubiquitination, are also involved in Akt activation. In this proposal, we have identified TRAF6 as a potential Akt E3 ligase that induces Akt K63-linked polyubiquitination and is critical for Akt phosphorylation and activation. Our results reveal that Akt K63 polyubiquitination may represent a novel mechanism by which oncogenic Akt signaling is regulated and that this study may have important clinical implications for the treatment of cancer. The objective of this application, which is the next step in pursuit of that goal, is to determine the potential role of TRAF6 and Akt K63-linked polyubiquitination in cancer progression and metastasis. The central hypothesis of the application is that Akt K63-linked polyubiquitination induced by TRAF6 orchestrates Akt membrane localization and subsequent phosphorylation. Our central hypothesis has been formulated on the basis of our strong preliminary data. The rationale of the proposed research is that, once the important role of TRAF6 and Akt polyubiquitination is confirmed and established in cancer progression and metastasis, the better treatment or target for human cancers can be achieved. We plan to test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: Aim 1) To determine the molecular mechanism by which Akt polyubiquitination regulates Akt activation. Aim 2) To determine the molecular mechanism by which Akt ubiquitination and deubiquitination are regulated. Aim 3) To determine in vivo role of TRAF6 in tumorigenesis and metastasis. This proposal work is innovative, because it provides for the first time that TRAF6 is an E3 ligase for Akt and Akt K63-linked polyubiquitination orchestrate Akt phosphorylation and activation. With respected outcomes, the combination of work in this proposal is collectively expected to uncover the critical role of TRAF6 and Akt K63-linked polyubiquitination in cancer development and metastasis. Such results will have an important positive impact, because identification of the important role of TRAF6 and Akt K63-linked polyubiquitination in cancer progression and metastasis may therefore provide potentially therapeutic targets for cancer treatment. Our results will also fundamentally advance our current understandings of how oncogenic Akt signaling is regulated and may have important clinical implications. PUBLIC HEALTH RELEVANCE: Although the extensive studies have been made for the role of PI3K/Akt signaling in cancers, how Akt membrane localization and phosphorylation is regulated remains largely unknown. Our study reveals that a novel posttranslational modification is operated on Akt and that Akt K63-linked polyubiquitnation by E3 ligase TRAF6 may play important roles in Akt membrane localization, phosphorylation, and tumorigenesis. Understanding the mechanism by which Akt activation is regulated may not only provide a great insight into how the oncogenic Akt signaling is regulated, but also offer the important therapeutic implications for human cancers.